The long term goal if the proposed research is to understand the inhibitory synaptic transmission in the inner retina of the vertebrates. Emphasis will be on the bipolar cells that contain inhibitory neurotransmitter, GABA (GABA-IR) in the tiger salamander retina. The hypothesis to be addressed is: GABA containing bipolar cells are an origin of sustained inhibitory inputs to the ON and OFF ganglion cells thereby providing the anatomical substrate for push-pull modulation of ganglion cell responses. Morphological and electrophysiological techniques will be used on single cells. The specific aims of this project are: (1) characterized the morphological types of HRP injected and/or Golgi stained bipolars. Single bipolar cells in the superfused retinal slices will be strained either with HRP injection or a Golgi collinear method in the isolated retinas; morphology will be studied with camera lucida and a Eutectic Neuron Tracing system; (2) identify the neurotransmitter content (GABA, glutamate) of different morphological types of labeled bipolar cells. GABA and glutamate content of the stained cells will be determined by post-embed immunofluorescence double labelling method at the light microscope (EM) level. (3) study the synaptic input and output target (amacrine cell and/or ganglion cell, bipolar cell) of GABA-IR bipolar cell at the electron microscope (EM) level. Results will be compared to bipolar cells that contain glutamate; (4) determine the synaptic connectivity of GABA-IR bipolar cells and physiologically identified and HRP injected ON and OFF sustained ganglion cell will be OFF light responses will be recorded intracellularly and the ganglion cell will be subsequently stained with HRP. The GABAergic and glutamatergic synaptic input from bipolar cells to the HRP stained ganglion cell will be studied using a post-embed immunogold method at the EM level. The retinas of urodele amphibians including salamanders, have been used for over 25 years to study mechanisms of retinal processing. However, our knowledge of the synaptic organization of these retinas has not kept pace with the vaster amount of physiological and pharmacological data. It is this gap that the proposal is designed to address. Marc has proposed, on phylogenetic grounds, that the retinas of urodeles can serve as an appropriate model for mammalian retinas. I predict that this project on inhibitory bipolar cells of salamander retinas. I predict that this project on inhibitory bipolar cells of salamander retina will have important implications for studies of inhibitory bipolar cells in rabbit, cat and by extension, primate retinas.